Catabolic Intermediates Research Articles

Catabolism of 4‐hydroxy‐2(E)‐nonenal (HNE) via omega oxidation in perfused rat livers

Oxidative stress triggers peroxidation of polyunsaturated fatty acids (FA) to HNE. HNE detoxification is classically defined by conjugation reactions and excretion. However, we previously showed that HNE is also degraded through α/β FA oxidation by 2 parallel pathways via the carboxyl end of its metabolite, 4‐ hydroxynonanoate (HNA); a process which is inhibited by increased FA concentrations. In this study, other catabolic intermediates were identified in perfused hearts and livers by gas chromatography and mass spectrometry. Perfusion with [2H11]HNE or [3,413C2]HNA revealed intermediates (4‐hydroxynonanedioyl‐ CoA, 4‐hydroxyheptanedioyl‐CoA and 4‐hydroxypentanedioyl‐ CoA) in the liver indicative of ω oxidation, implying HNE catabolism can proceed from either end of the molecule. In hearts perfused with labeled HNE or HNA catabolism by α/β FA oxidation was evident, however, ω oxidation of HNE was not detected. The lack of HNE ω oxidation in the heart could play a role in its susceptibility to oxidant stress, particularly in pathological conditions such as diabetes and heart failure where alterations in FA metabolism and oxidative damage occur. These alternative detoxification pathways not only expand the understanding of how this endogenous toxicant is handled in the cell, but also suggest a novel interplay between energy metabolism and cellular antioxidant defenses. (Supported by NIH, NSF, AHA & CWRU)

Fumarylacetoacetate inhibits the initial step of the base excision repair pathway: implication for the pathogenesis of tyrosinemia type I

Hereditary tyrosinemia type I (HT1) is an autosomal recessive disease caused by a deficiency in human fumarylacetoacetate (FAA) hydrolase (FAH), which is the last enzyme in the catabolic pathway of tyrosine. Several reports suggest that intracellular accumulation of intermediates of tyrosine catabolism, such as FAA and succinylacetone (SA) is important for the pathogenesis in liver and kidney of HT1 patients. In this work, we examined the effect of FAA and SA on DNA glycosylases initiating base excision repair (BER), which is the most important pathway for removing mutagenic DNA base lesions. In vitro assays monitoring DNA glycosylase activities demonstrated that FAA but not SA inhibited base removal. In particular, the Neil1 and Neil2 DNA glycosylases were strongly inhibited, whereas inhibition of Nth1 and Ogg1 were less efficient. These DNA glycosylases initiate excision of a broad range of mutagenic oxidative base lesions. Further, FAA showed a modest inhibitory effect on the activity of the alkylbase DNA glycosylase Aag and no significant inhibition of the uracil DNA glycosylase Ung2. These data indicate that FAA inhibition of DNA glycosylases removing oxidative base lesions in HT1 patients may increase mutagenesis, suggesting an important mechanism for development of hepatocarcinoma and somatic mosaicism.

Botrytis cinerea mutants deficient in D-galacturonic acid catabolism have a perturbed virulence on Nicotiana benthamiana and Arabidopsis, but not on tomato.

D-Galacturonic acid is the most abundant monosaccharide component of pectic polysaccharides that comprise a significant part of most plant cell walls. Therefore, it is potentially an important nutritional factor for Botrytis cinerea when it grows in and through plant cell walls. The d-galacturonic acid catabolic pathway in B. cinerea consists of three catalytic steps converting d-galacturonic acid to pyruvate and l-glyceraldehyde, involving two nonhomologous galacturonate reductase genes (Bcgar1 and Bcgar2), a galactonate dehydratase gene (Bclgd1) and a 2-keto-3-deoxy-l-galactonate aldolase gene (Bclga1). Knockout mutants in each step of the pathway (ΔBcgar1/ΔBcgar2, ΔBclgd1 and ΔBclga1) showed reduced virulence on Nicotiana benthamiana and Arabidopsis thaliana leaves, but not on Solanum lycopersicum leaves. The cell walls of N. benthamiana and A. thaliana leaves were shown to have a higher d-galacturonic acid content relative to those of S. lycopersicum. The observation that mutants displayed a reduction in virulence, especially on plants with a high d-galacturonic acid content in the cell walls, suggests that, in these hosts, d-galacturonic acid has an important role as a carbon nutrient for B. cinerea. However, additional in vitro growth assays with the knockout mutants revealed that B. cinerea growth is reduced when d-galacturonic acid catabolic intermediates cannot proceed through the entire pathway, even when fructose is present as the major, alternative carbon source. These data suggest that the reduced virulence of d-galacturonic acid catabolism-deficient mutants on N. benthamiana and A. thaliana is not only a result of the inability of the mutants to utilize an abundant carbon source as nutrient, but also a result of the growth inhibition by catabolic intermediates.

The Steroid Catabolic Pathway of the Intracellular Pathogen Rhodococcus equi Is Important for Pathogenesis and a Target for Vaccine Development

Rhodococcus equi causes fatal pyogranulomatous pneumonia in foals and immunocompromised animals and humans. Despite its importance, there is currently no effective vaccine against the disease. The actinobacteria R. equi and the human pathogen Mycobacterium tuberculosis are related, and both cause pulmonary diseases. Recently, we have shown that essential steps in the cholesterol catabolic pathway are involved in the pathogenicity of M. tuberculosis. Bioinformatic analysis revealed the presence of a similar cholesterol catabolic gene cluster in R. equi. Orthologs of predicted M. tuberculosis virulence genes located within this cluster, i.e. ipdA (rv3551), ipdB (rv3552), fadA6 and fadE30, were identified in R. equi RE1 and inactivated. The ipdA and ipdB genes of R. equi RE1 appear to constitute the α-subunit and β-subunit, respectively, of a heterodimeric coenzyme A transferase. Mutant strains RE1ΔipdAB and RE1ΔfadE30, but not RE1ΔfadA6, were impaired in growth on the steroid catabolic pathway intermediates 4-androstene-3,17-dione (AD) and 3aα-H-4α(3′-propionic acid)-5α-hydroxy-7aβ-methylhexahydro-1-indanone (5α-hydroxy-methylhexahydro-1-indanone propionate; 5OH-HIP). Interestingly, RE1ΔipdAB and RE1ΔfadE30, but not RE1ΔfadA6, also displayed an attenuated phenotype in a macrophage infection assay. Gene products important for growth on 5OH-HIP, as part of the steroid catabolic pathway, thus appear to act as factors involved in the pathogenicity of R. equi. Challenge experiments showed that RE1ΔipdAB could be safely administered intratracheally to 2 to 5 week-old foals and oral immunization of foals even elicited a substantial protective immunity against a virulent R. equi strain. Our data show that genes involved in steroid catabolism are promising targets for the development of a live-attenuated vaccine against R. equi infections.

Equilibrative nucleoside transporter 1 (ENT1) is critical for pollen germination and vegetative growth in Arabidopsis

ENT1 of Arabidopsis thaliana was the first member of the equilibrative nucleoside transporter (ENT) family to be identified in plants and characterized as a cellular, high-affinity nucleoside importer. Evidence is presented here for a tonoplast localization of ENT1 based on proteome data and Western blot analyses. Increased export of adenosine from reconstituted tonoplast preparations from 35S:ENT1 mutants compared with those from the wild type and ENT1-RNAi mutants support this view. Furthermore, increased vacuolar adenosine and vacuolar 2′3′-cAMP (an intermediate of RNA catabolism) contents in ENT1-RNAi mutants, but decreased contents of these metabolites in 35S:ENT1 over-expresser mutants, were observed. An up-regulation of the salvage pathway was detected in the latter mutants, leading to the conclusion that draining the vacuolar adenosine storage by ENT1 over-expression interferes with cellular nucleotide metabolism. As a consequence of the observed metabolic alterations 35S:ENT1 over-expresser mutants exhibited a smaller phenotypic appearance compared with wild-type plants. In addition, ENT1:RNAi mutants exhibited significantly lower in vitro germination of pollen and contained reduced internal and external ATP levels. This indicates that ENT1-mediated nucleosides, especially adenosine transport, is important for nucleotide metabolism, thus influencing growth and pollen germination.

Strict and direct transcriptional repression of the pobA gene by benzoate avoids 4‐hydroxybenzoate degradation in the pollutant degrader bacterium Cupriavidus necator JMP134

As other environmental bacteria, Cupriavidus necator JMP134 uses benzoate as preferred substrate in mixtures with 4-hydroxybenzoate, strongly inhibiting its degradation. The mechanism underlying this hierarchical use was studied. A C. necator benA mutant, defective in the first step of benzoate degradation, is unable to metabolize 4-hydroxybenzoate when benzoate is also included in the medium, indicating that this substrate and not one of its catabolic intermediates is directly triggering repression. Reverse transcription polymerase chain reaction analysis revealed that 4-hydroxybenzoate 3-hydroxylase-encoding pobA transcripts are nearly absent in presence of benzoate and a fusion of pobA promoter to lacZ reporter confirmed that benzoate drastically decreases the transcription of this gene. Expression of pobA driven by a heterologous promoter in C. necator benA mutant, allows growth on 4-hydroxybenzoate in presence of benzoate, overcoming its repressive effect. In contrast with other bacteria, regulators of benzoate catabolism do not participate in repression of 4-hydroxybenzoate degradation. Moreover, the effect of benzoate on pobA promoter can be observed in heterologous strains with the sole presence of PobR, the transcriptional activator of pobA gene, indicating that PobR is enough to fully reproduce the phenomenon. This novel mechanism for benzoate repression is probably mediated by direct action of benzoate over PobR.

Mechanisms of oxysterol-induced carcinogenesis

Oxysterols are oxidation products of cholesterol that are generated by enzymatic reactions mediated by cytochrome P450 family enzymes or by non-enzymatic reactions involving reactive oxygen and nitrogen species. Oxysterols play various regulatory roles in normal cellular processes such as cholesterol homeostasis by acting as intermediates in cholesterol catabolism. Pathological effects of oxysterols have also been described, and various reports have implicated oxysterols in several disease states, including atherosclerosis, neurological disease, and cancer. Numerous studies show that oxysterols are associated with various types of cancer, including cancers of the colon, lung, skin, breast and bile ducts. The molecular mechanisms whereby oxysterols contribute to the initiation and progression of cancer are an area of active investigation. This review focuses on the current state of knowledge regarding the role of oxysterols in carcinogenesis. Mutagenicity of oxysterols has been described in both nuclear and mitochondrial DNA. Certain oxysterols such as cholesterol-epoxide and cholestanetriol have been shown to be mutagenic and genotoxic. Oxysterols possess pro-oxidative and pro-inflammatory properties that can contribute to carcinogenesis. Oxysterols can induce the production of inflammatory cytokines such as interleukin-8 and interleukin-1β. Certain oxysterols are also involved in the induction of cyclo-oxygenase-2 expression. Inflammatory effects can also be mediated through the activation of liver-X-receptor, a nuclear receptor for oxysterols. Thus, several distinct molecular mechanisms have been described showing that oxysterols contribute to the initiation and progression of cancers arising in various organ systems.

Gluconate Metabolism Is Required for Virulence of the Soft-Rot Pathogen Pectobacterium carotovorum

Pectobacterium carotovorum is a ubiquitous soft rot pathogen that uses global virulence regulators to coordinate pathogenesis in response to undefined environmental conditions. We characterize an operon in P. carotovorum required for gluconate metabolism and virulence. The operon contains four genes that are highly conserved among proteobacteria (initially annotated ygbJKLM), one of which was misassigned as a type III secreted effector, (ygbK, originally known as hopAN1). A mutant with a deletion-insertion within this operon is unable to metabolize gluconate, a precursor for the pentose phosphate pathway. The mutant exhibits attenuated growth on the leaves of its host of isolation, potato, and those of Arabidopsis thaliana. Notably, the mutant hypermacerates potato tubers and is deficient in motility. Global virulence regulators that are responsive to cell wall pectin breakdown products and other undefined environmental signals, KdgR and FlhD, respectively, are misregulated in the mutant. The alteration of virulence mediated via changes in transcription of known global virulence regulators in our ygbJ-M operon mutant suggests a role for host-derived catabolic intermediates in P. carotovorum pathogenesis. Thus, we rename this operon in P. carotovorum vguABCD for virulence and gluconate metabolism.

Abstract 58: Hyaluronan metabolism enhances the Warburg effect and provides an alternate energy source in the breast cancer microenvironment

Abstract Introduction: A hallmark of cancer is a shift in cellular metabolism away from oxidative phosphorylation of glucose towards aerobic glycolysis thereby facilitating the rapid generation of energy required for proliferation. In breast cancer, the overproduction of hyaluronan (HA) is associated with poor disease outcomes where its metabolism is a finely regulated equilibrium between rapid synthesis and degradation. Synthetic products are involved in the structural integrity of the tumoral architecture while catabolic intermediates contribute to cell proliferation, neovascularisation and metastasis but little is known about their role in the generation of intratumoral energy. Methods: A hyaluronanhigh producing cell line was generated by the stable transfection of HAS2 into the ZR-75-1 breast cancer cell line. The HA metabolism and expression of the HA receptor, CD44 was characterised in the parental, vector- and HAS2-transfected cell lines. The effect of elevated HA synthesis on the glycolytic phenotype was determined by quantitation of phosphoinositide 3-kinase/Akt pathway activation, transketolase-like protein 1 (TKTL1), pyruvate kinase M2 (PKM2), glucose transporter 1 (GLUT1), aerobic glycolysis, cell proliferation and metastasis both in vitro and in vivo. Results: Compared to the controls, HAS2-transfected cells liberated up to 10-fold more HA and had increased expression of CD44s and CD44v9. The resultant increase in CD44-HA interactions mediated in vitro activation of the PI3K/Akt pathway, enhanced proliferation and metastatic capacity, increased propensity for aerobic glycolysis and a higher HA metabolic index. High levels of HA production conferred a characteristic glycolytic phenotype to HAS2 transfectants where Akt and TKTL1 were up-regulated and PKM2 reduced. Unlike the classic glycolytic phenotype, GLUT1 was not increased, but down-regulated 3-fold in the HAS2 cells. HA-induced enhancement of the glycolytic phenotype was confirmed in vivo by a >6 fold increase in endpoint tumor volume and reduced overall survival in an animal model of metastases. Conclusions: Elevated HA production confers an aggressive phenotype to breast cancer where CD44-HA interactions alter the metabolic fate of the cell driving it into an aerobic glycolytic pathway. This study has provided preliminary evidence that in highly proliferative breast cancer glucose may not be the sole catabolic substrate and hyaluronan may contribute to this process. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 58.

Electron Transport Chain-dependent and -independent Mechanisms of Mitochondrial H2O2 Emission during Long-chain Fatty Acid Oxidation

Oxidative stress in skeletal muscle is a hallmark of various pathophysiologic states that also feature increased reliance on long-chain fatty acid (LCFA) substrate, such as insulin resistance and exercise. However, little is known about the mechanistic basis of the LCFA-induced reactive oxygen species (ROS) burden in intact mitochondria, and elucidation of this mechanistic basis was the goal of this study. Specific aims were to determine the extent to which LCFA catabolism is associated with ROS production and to gain mechanistic insights into the associated ROS production. Because intermediates and by-products of LCFA catabolism may interfere with antioxidant mechanisms, we predicted that ROS formation during LCFA catabolism reflects a complex process involving multiple sites of ROS production as well as modified mitochondrial function. Thus, we utilized several complementary approaches to probe the underlying mechanism(s). Using skeletal muscle mitochondria, our findings indicate that even a low supply of LCFA is associated with ROS formation in excess of that generated by NADH-linked substrates. Moreover, ROS production was evident across the physiologic range of membrane potential and was relatively insensitive to membrane potential changes. Determinations of topology and membrane potential as well as use of inhibitors revealed complex III and the electron transfer flavoprotein (ETF) and ETF-oxidoreductase, as likely sites of ROS production. Finally, ROS production was sensitive to matrix levels of LCFA catabolic intermediates, indicating that mitochondrial export of LCFA catabolic intermediates can play a role in determining ROS levels.

Macrophages, Oxysterols and Atherosclerosis

Macrophages play central roles in immunity and homeostasis, and contribute to the pathogenesis of atherosclerosis through their accumulation of cholesterol and the production of inflammatory mediators and cytokines. Recent studies indicate that oxysterols influence diverse aspects of macrophage biology. Rather than simply being intermediates of cholesterol catabolism, oxysterols are also potent bioactive lipids that regulate lipid metabolism, immune function, and cytotoxicity. These functions are mediated by specific oxysterol sensors, including liver X receptors (LXR), Insigs, and members of the oxysterol binding protein (OSBP) and OSBP-related protein family. The mechanisms of oxysterol-induced functions and their physiological roles in macrophages are reviewed.

The Behavior and Significance of Degradative Plasmids Belonging to Inc Groups in <i>Pseudomonas</i> within Natural Environments and Microcosms

Over the past few decades, degradative plasmids have been isolated from bacteria capable of degrading a variety of both natural and man-made compounds. Degradative plasmids belonging to three incompatibility (Inc) groups in Pseudomonas (IncP-1, P-7, and P-9) have been well studied in terms of their replication, maintenance, and capacity for conjugative transfer. The host ranges of these plasmids are determined by replication or conjugative transfer systems. The host range of IncP-1 is broad, that of IncP-9 is intermediate, and that of IncP-7 is narrow. To understand the behavior of these plasmids and their hosts in various environments, the survivability of inocula, stability or transferability, and efficiency of biodegradation in environments and microcosms have been monitored. The biodegradation and plasmid transfer in various environments have been observed for all three groups, although the kinds of transconjugants differed with the Inc groups. In some cases, the deletion and amplification of catabolic genes acted to reduce the production of toxic catabolic intermediates, or to increase the activity on a particular catabolic pathway. The combination of degradative genes, the plasmid backbone of each Inc group, and the host of the plasmids is key to the degraders adapting to various hosts or to heterogeneous environments.

Differential Membrane Proteome Analysis Reveals Novel Proteins Involved in the Degradation of Aromatic Compounds in Geobacter metallireducens

Aromatic compounds comprise a large class of natural and man-made compounds, many of which are of considerable concern for the environment and human health. In aromatic compound-degrading anaerobic bacteria the central intermediate of aromatic catabolism, benzoyl coenzyme A, is attacked by dearomatizing benzoyl-CoA reductases (BCRs). An ATP-dependent BCR has been characterized in facultative anaerobes. In contrast, a previous analysis of the soluble proteome from the obligately anaerobic model organism Geobacter metallireducens identified genes putatively coding for a completely different dearomatizing BCR. The corresponding BamBCDEFGHI complex is predicted to comprise soluble molybdenum or tungsten, selenocysteine, and FeS cluster-containing components. To elucidate key processes involved in the degradation of aromatic compounds in obligately anaerobic bacteria, differential membrane protein abundance levels from G. metallireducens grown on benzoate and acetate were determined by the MS-based spectral counting approach. A total of 931 proteins were identified by combining one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis with liquid chromatography-tandem mass spectrometry. Several membrane-associated proteins involved in the degradation of aromatic compounds were newly identified including proteins with similarities to modules of NiFe/heme b-containing and energy-converting hydrogenases, cytochrome bd oxidases, dissimilatory nitrate reductases, and a tungstate ATP-binding cassette transporter system. The transcriptional regulation of differentially expressed genes was analyzed by quantitative reverse transcription-PCR; in addition benzoate-induced in vitro activities of hydrogenase and nitrate reductase were determined. The results obtained provide novel insights into the poorly understood degradation of aromatic compounds in obligately anaerobic bacteria.

Light-Independent Cell Death Induced by Accumulation of Pheophorbide a in Arabidopsis thaliana

Tetrapyrroles are well-known photosensitizers. In plants, various intermediate molecules of tetrapyrrole metabolism have been reported to induce cell death in a light-dependent manner. In contrast to these reports, we found that pheophorbide a, a key intermediate of chlorophyll catabolism, causes cell death in complete darkness in a transgenic Arabidopsis plant, As-ACD1. In this plant, expression of mRNA for pheophorbide a oxygenase was suppressed by expression of Acd1 antisense RNA; thus, As-ACD1 accumulated an excessive amount of pheophorbide a when chlorophyll breakdown occurred. We observed that when senescence was induced by a continuous dark period, leaves of As-ACD1 plants became dehydrated. By measuring electrolyte leakage, we estimated that >50% of the leaf cells underwent cell death within a 5 d period of darkness. Light and electron microscopic observations indicated that the cellular structure had collapsed in a large population of cells. Partially covering a leaf with aluminum foil resulted in light-independent cell death in the covered region and induced bleaching in the uncovered regions. These results indicate that accumulation of pheophorbide a induces cell death under both darkness and illumination, but the mechanisms of cell death under these conditions may differ. We discuss the possible mechanism of light-independent cell death and the involvement of pheophorbide a in the signaling pathway for programmed cell death.

Establishment of Oxidative d -Xylose Metabolism in Pseudomonas putida S12

The oxidative D-xylose catabolic pathway of Caulobacter crescentus, encoded by the xylXABCD operon, was expressed in the gram-negative bacterium Pseudomonas putida S12. This engineered transformant strain was able to grow on D-xylose as a sole carbon source with a biomass yield of 53% (based on g [dry weight] g D-xylose(-1)) and a maximum growth rate of 0.21 h(-1). Remarkably, most of the genes of the xylXABCD operon appeared to be dispensable for growth on D-xylose. Only the xylD gene, encoding D-xylonate dehydratase, proved to be essential for establishing an oxidative D-xylose catabolic pathway in P. putida S12. The growth performance on D-xylose was, however, greatly improved by coexpression of xylXA, encoding 2-keto-3-deoxy-D-xylonate dehydratase and alpha-ketoglutaric semialdehyde dehydrogenase, respectively. The endogenous periplasmic glucose dehydrogenase (Gcd) of P. putida S12 was found to play a key role in efficient oxidative D-xylose utilization. Gcd activity not only contributes to D-xylose oxidation but also prevents the intracellular accumulation of toxic catabolic intermediates which delays or even eliminates growth on D-xylose.

Effects of temperature and feed strength on a carrier anaerobic baffled reactor treating dilute wastewater

A carrier anaerobic baffled reactor (CABR), containing hollow-sphere bamboo carriers, was used to examine theeffects of organic and temperature shock loads for a constant hydraulic retention time. The relationships among themacroscopic observables (pH, COD and SS), catabolic intermediates (VFA) and microcosm alternation (TTC–DHA)were studied. A slight fluctuation in pH was observed, ranging from 6.5 to 7.5 throughout the experiments. TheCABR had a COD removal efficiency of 91% with an effluent suspended solids (SS) of 15 mg L −1 at 28°C and a feedstrength of 600 mg L −1 chemical oxygen demand (COD). The reactor could run steadily without souring due to thevolatile fatty acids (VFA) production (<150 mg L −1) and low VFA/alkalinity ratio (<0.23), although the effluent VFAconcentration increased with decreasing operational temperature. For a substrate low in nutrient, the microorganismsconverted the limited substrate into acetate to supply the essential nicotinamide adenine dinucleotide for cellularsynthesis. The tetrazolium chloride–dehydrogenase activity (TTC–DHA) of the attached biomass was higher thanthat of flock biomass at low feed concentration, whereas the result was the opposite at high feed concentration.Microorganisms with lower activity but higher shock resistance were dominant in the first chamber. Therefore, theCABR had high shock resistance and adaptability irrespective of temperature and feed strength.

Phenylalanine-induced leucopenia in genetic and dichloroacetic acid generated deficiency of glutathione transferase Zeta

Glutathione transferase Zeta (GSTZ1-1) is identical to maleylacetoacetate isomerase and catalyses a significant step in the catabolism of phenylalanine and tyrosine. Exposure of GSTZ1-1 deficient mice to high dietary phenylalanine causes a rapid loss of circulating white blood cells (WBCs). The loss was significant ( P < 0.05) after 2 days and total WBCs were reduced by 60% after 6 days. The rapid loss of WBCs was attributed to the accumulation of the catabolic intermediates maleylacetoacetate or maleylacetone (MA) in the circulation. Serum from GSTZ1-1 deficient mice treated with phenylalanine was cytotoxic to splenocytes from normal BALB/c mice and direct incubation of normal splenocytes with MA caused a rapid loss of viability. Dichloroacetic acid (DCA) has been used therapeutically to treat lactic acidosis and is potentially of use in cancer chemotherapy. Since DCA can inactivate GSTZ1-1 there is a possibility that long-term treatment of patients with DCA could cause GSTZ1-1 deficiency and susceptibility to oxidative stress and phenylalanine/tyrosine-induced WBC loss. However, although we found that DCA at 200 mg/(kg day) causes a severe loss of hepatic GSTZ1-1 activity in BALB/c mice, it did not induce WBC cytotoxicity when combined with high dietary phenylalanine.

Biodegradation of methyl parathion and p-nitrophenol: evidence for the presence of a p-nitrophenol 2-hydroxylase in a Gram-negative Serratia sp. strain DS001

A soil bacterium capable of utilizing methyl parathion as sole carbon and energy source was isolated by selective enrichment on minimal medium containing methyl parathion. The strain was identified as belonging to the genus Serratia based on a phylogram constructed using the complete sequence of the 16S rRNA. Serratia sp. strain DS001 utilized methyl parathion, p-nitrophenol, 4-nitrocatechol, and 1,2,4-benzenetriol as sole carbon and energy sources but could not grow using hydroquinone as a source of carbon. p-Nitrophenol and dimethylthiophosphoric acid were found to be the major degradation products of methyl parathion. Growth on p-nitrophenol led to release of stoichiometric amounts of nitrite and to the formation of 4-nitrocatechol and benzenetriol. When these catabolic intermediates of p-nitrophenol were added to resting cells of Serratia sp. strain DS001 oxygen consumption was detected whereas no oxygen consumption was apparent when hydroquinone was added to the resting cells suggesting that it is not part of the p-nitrophenol degradation pathway. Key enzymes involved in degradation of methyl parathion and in conversion of p-nitrophenol to 4-nitrocatechol, namely parathion hydrolase and p-nitrophenol hydroxylase component "A" were detected in the proteomes of the methyl parathion and p-nitrophenol grown cultures, respectively. These studies report for the first time the existence of a p-nitrophenol hydroxylase component "A", typically found in Gram-positive bacteria, in a Gram-negative strain of the genus Serratia.

Lipoprotein distribution and biological variation of 24S- and 27-hydroxycholesterol in healthy volunteers

24S- and 27-hydroxycholesterol are obligatory intermediates of cholesterol catabolism and play an important role in the maintenance of whole-body cholesterol homeostasis. Using an HPLC–MS method for oxysterol quantification, the distribution of esterified and unesterified oxysterols in lipoprotein subfractions as well as the influence of daytime, food intake and menstrual cycle on oxysterol concentrations were investigated in healthy volunteers. Moreover, reference intervals for 24S- and 27-hydroxycholesterol in plasma as well as the corresponding levels for 27-hydroxycholesterol in the HDL subfraction were established in 100 healthy volunteers. Both circulating oxysterols are mainly transported in association with HDL and LDL – primarily in the esterified form. No significant diurnal changes and no variations during menstrual cycle of either absolute or cholesterol-related plasma levels were detected. In contrast to 24S-hydroxycholesterol in plasma and 27-hydroxycholesterol in the HDL subfraction, the 95% reference intervals of 27-hydroxycholesterol both in plasma and the non-HDL subfraction were higher in males than in females. The concentrations of 27-hydroxycholesterol in plasma and the non-HDL subfraction showed strong positive correlations with the concentrations of cholesterol, non-HDL cholesterol and triglycerides. Our data on the lipoprotein distribution of oxysterols as well as on their intra- and inter-individual variation set the stage for future clinical studies.

Structures of Biologically Active Oxysterols Determine Their Differential Effects on Phospholipid Membranes

Oxysterols, derivatives of cholesterol that contain a second oxygen moiety, are intermediates in cholesterol catabolism, regulators of lipid metabolism, and toxic sterols with proatherogenic effects. In model membranes, cholesterol and eight selected oxysterols were compared by fluorescence probe techniques that measure changes in bilayer order and phase behavior and by the formation of detergent-resistant membranes (DRM). The oxysterols were modified on the sterol nucleus or on the isooctyl side chain. The model membranes consisted of dipalmitoyl phosphatidylcholine (DPPC) and mixtures of dioleoyl phosphatidylcholine with DPPC and with sphingomyelin. The different oxysterols induced changes in membrane properties according to the differences in their structures. Whereas the effects of some oxysterols on membrane order, fluorescence probe microenvironment, and DRM formation were similar to those of cholesterol, others had little or no effect. An empirical correlation ranking the oxysterols by their ability to modify membrane biophysical properties when compared to cholesterol led to a significant structure/function relationship between the biophysical measurements and an important cellular phenomenon, apoptosis. 7beta-Hydroxycholesterol, which is the most cytotoxic of the eight selected oxysterols, was one of the least cholesterol-like with respect to modification of membrane properties. The results suggest that an underlying mechanism for oxysterol-induced apoptosis in cells, e.g., monocyte/macrophages, should include their biophysical effects on membranes, such as the regulation of the formation and composition of sterol-rich membrane domains.